Clostridium histolyticum enzymes and methods for the use thereof

ABSTRACT

The invention relates to recombinant nucleic acid and polypeptides encoding collagenase I and collagenase II, methods for the preparation thereof and methods for the use thereof. The invention also encompasses methods related to releasing a composition comprising collagenase prior to therapeutic administration.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/328,772 filed on Jul. 11, 2014, now U.S. Pat. No. 9,757,435, which isa continuation application of International Application No.PCT/US13/020940, which designated the United States and was filed onJan. 10, 2013, published in English which claims the benefit of U.S.Provisional Application No. 61/585,909 filed Jan. 12, 2012. The entirecontents of the above-referenced applications are incorporated byreference herein.

BACKGROUND OF THE INVENTION

Collagen is the major structural constituent of mammalian organisms andmakes up a large portion of the total protein content of skin and otherparts of the animal body. In humans, it is particularly important in theprocesses of wound healing process and natural aging. Various skintraumas, including burns, surgery, and infection, are characterized bythe accumulation of fibrous tissue rich in collagen and having increasedproteoglycan content. In addition to the replacement of the normaltissue which has been damaged or destroyed, excessive and disfiguringdeposits of new tissue sometimes form during the healing process. Theexcess collagen deposition has been attributed to a disturbance in thebalance between collagen synthesis and collagen degradation.

Diseases and conditions associated with excess collagen deposition andthe erratic accumulation of fibrous tissue rich in collagen can bereferred to as “collagen-mediated diseases.” Collagenase, an enzyme thathas the specific ability to digest collagen, has been used to treat avariety of collagen-mediated diseases, including, for example,Dupuytren's contracture, Peyronie's disease, lipoma and adhesivecapsulitis. U.S. Pat. Nos. 6,086,872 and 5,589,171, incorporated hereinby reference, disclose the use of collagenase preparations in thetreatment of Dupuytren's disease. U.S. Pat. No. 6,022,539, incorporatedherein by reference, discloses the use of collagenase preparations inthe treatment of Peyronie's disease. U.S. Pat. Nos. 6,958,150 and7,842,673, incorporated herein by reference, disclose the use ofcollagenase for the treatment of lipoma. U.S. Patent ApplicationPublication No. 2006/020448A1, incorporated herein by reference,discloses the use of collagenase in the treatment of adhesivecapsulitis. Collagenase for use in therapy may be obtained from avariety of sources including mammalian, fungal, and bacterial sources.One common source of crude collagenase is from a bacterial fermentationprocess, specifically the fermentation of Clostridium histolyticum (C.histolyticum). The crude collagenase obtained from C. histolyticum maybe purified using any of a number of chromatographic techniques.

One drawback of the fermentation of bacteria is that various toxins willbe produced, that if present in the therapeutic composition, would bedetrimental to the health of the patient. For example, C. histolyticumfermentation results in the synthesis of the hemolytic toxins alpha andepsilon, which can cause lysis of red blood cells (hemolysis),potentially leading to hemolytic crisis and hemolytic anemia. Hemolyticcrisis occurs when there is a rapid destruction of large numbers of redblood cells in conjunction with the body's inability to replenish thered blood cells quickly enough to reestablish normal red blood celllevels. A hemolytic crisis causes acute (and often severe) hemolyticanemia, and can result in fatigue, shortness of breath, dizziness,headache, coldness in the hands and feet, pale skin, chest pain,jaundice, pain in the upper abdomen, leg ulcers and pain, severereactions to a blood transfusion, arrhythmias, an enlarged heart, andheart failure. In order to ensure that the therapeutic collagenasepreparation does not contain hemolytic toxins that might be expressedduring C. histolyticum fermentation, a method for releasing a drugproduct prior to administration to a patient is presented.

As discussed above, collagenase for use in therapy can be obtained froma variety of sources such as bacterial sources (e.g. from thefermentation of C. histolyticum). It would be useful to developadditional sources of collagenase such as recombinant forms ofcollagenase enzymes.

SUMMARY OF THE INVENTION

In some aspects, the present invention is based on the discovery ofmutated polynucleotide sequences that encode functional collagenase Iand collagenase II. The invention thus encompasses recombinant nucleicacid and polypeptides comprising the novel polynucleotide or polypeptidesequences and methods for the use thereof. The present invention alsoprovides a method for detecting the secretion of a hemolytic toxin by abacterial production strain, wherein the production strain produces acollagenase, prior to therapeutic administration of said collagenase toa patient and methods for detecting the presence of a hemolytic toxin ina collagenase composition.

In one embodiment, the invention is directed to a recombinant nucleicacid molecule comprising a polynucleotide having the sequence of SEQ IDNO: 1 (collagenase I nucleotide sequence) or the complement of SEQ IDNO: 1. In certain aspects, the recombinant nucleic acid furthercomprises a heterologous regulatory sequence operably linked to thepolynucleotide. In certain additional embodiments, the invention is arecombinant nucleic acid molecule consisting of a polynucleotide of SEQID NO: 1. In yet additional aspects, the invention relates to arecombinant nucleic acid molecule consisting of a polynucleotide of SEQID NO: 1 and a heterologous regulator sequence operably linked to thepolynucleotide.

In another embodiment, the invention is a recombinant nucleic acidmolecule comprising a polynucleotide having the sequence of SEQ ID NO: 2(collagenase II nucleotide sequence) or the complement of SEQ ID NO: 2.In certain aspects, the recombinant nucleic acid further comprises aheterologous promoter operatively linked to the polynucleotide. Incertain additional embodiments, the invention is a recombinant nucleicacid molecule consisting of a polynucleotide of SEQ ID NO: 2. In yetadditional aspects, the invention relates to a recombinant nucleic acidmolecule consisting of a polynucleotide of SEQ ID NO: 2 and aheterologous regulator sequence operably linked to the polynucleotide.

The invention also includes recombinant polypeptides encoded by arecombinant nucleic acid comprising a polynucleotide having the sequenceof SEQ ID NO: 1 or SEQ ID NO: 2.

In certain additional embodiments, the invention is directed to anexpression cassette comprising a recombinant nucleic acid, wherein thenucleic acid comprises a polynucleotide having the sequence of SEQ IDNO: 1 or SEQ ID NO: 2.

In yet an additional embodiment, the invention is directed to a vectorcomprising a recombinant nucleic acid, wherein the nucleic acidcomprises a polynucleotide having the sequence of SEQ ID NO: 1 or SEQ IDNO: 2. In some embodiments, the vector is a plasmid.

In a further aspect, the invention is directed to a recombinant hostcell comprising the vector or plasmid comprising a polynucleotide havingthe sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The invention alsoencompasses a method of producing collagenase I or collagenase IIcomprising culturing the host cell under conditions suitable forexpression of the nucleic acid and recovering the collagenase I orcollagenase II. The invention also includes a collagenase enzymeproduced by culturing the recombinant host cell.

In some embodiments, the invention is directed to a recombinantlyproduced collagenase I comprising the amino acid sequence of SEQ ID NO:3, a recombinantly produced collagenase II comprising the amino acidsequence SEQ ID NO: 4, a recombinantly produced collagenase I comprisingthe amino acid sequence of SEQ ID NO: 5, or a recombinantly producedcollagenase II comprising the amino acid sequence of SEQ ID NO: 6.

Also included in the present invention are pharmaceutical compositionscomprising collagenase I as described herein, collagenase II asdescribed herein, or a combination thereof. In certain aspects, thepresent invention is directed to a pharmaceutical composition comprisinga pharmaceutically acceptable carrier and a polypeptide comprising theamino sequence of SEQ ID NO: 3, a polypeptide comprising the amino acidsequence of SEQ ID NO: 4, or a combination thereof. In certainadditional aspects, the present invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a polypeptide comprising the amino sequence of SEQ ID NO: 5,a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, or acombination thereof. The invention additionally includes methods oftreating a collagen-mediated disease comprising administering aneffective amount of collagenase I, collagenase II, or a combinationthereof.

As discussed above, the invention encompasses methods for detecting thesecretion of a hemolytic toxin by a bacterial production strain andmethods for detecting the presence of a hemolytic toxin in a collagenasecomposition.

In one embodiment of the invention, a bacterial strain that producescollagenase is tested for the production of hemolytic toxins using ahemolysis assay. In one aspect, the hemolysis assay is performed using ablood agar substrate.

In another embodiment, a collagenase product is tested for the presenceof hemolytic toxins using a hemolysis assay. In certain aspects, thehemolysis assay is performed using a blood agar substrate. In additionalaspects, the hemolysis assay is performed using photometric detection ofreleased hemoglobin. The absence of hemolytic toxins, as determined by ahemolysis assay or photometric detection, would support the release ofthe drug product for therapeutic administration.

Various strains of collagenase-producing bacteria can be assayed forhemolytic activity according to a method of the invention, in support ofthe release of a collagenase drug product for therapeuticadministration. For example, members of the genera Actinobacillus,Actinomadura, Bacillus, Bacteroides, Bifidobacterium, Brucella,Capnocytophaga, Clostridium, Enterococcus, Escherichia, Eubacterium,Flavobacterium, Fusobacterium, Peptococcus, Peptostreptococcus,Porphyromonas, Prevotella, Proteus, Pseudomonas, Serratia,Staphylococcus, Streptomyces, Streptococcus, Treponema, and Vibrio canbe assayed for hemolytic activity according to a method of theinvention, in support of the release of a collagenase drug product fortherapeutic administration.

In another embodiment, a collagenase product produced by, and purifiedfrom, a strain of collagenase-producing bacteria is assayed forhemolytic activity according to a method of the invention, in support ofthe release of a collagenase drug product for therapeuticadministration. In some embodiments, the production strain is selectedfrom, but not limited to, the above-listed genera. In another aspect ofthe invention, the production strain is an Escherichia coli (E. coli)strain, including forms of E. coli that have been transformed withrecombinant forms of collagenase I and collagenase II. In certainaspects of the invention, the production strain is a Clostridiumperfringens (C. prefrigens) strain. In additional aspects, theproduction strain is a C. histolyticum strain.

In yet another embodiment of the invention, the collagenase compositionis assayed for hemolytic activity according to a method of theinvention, wherein the collagenase composition comprises a combinationof purified C. histolyticum collagenase I and collagenase II. In anadditional embodiment, the invention is a method of producing a drugproduct consisting of C. histolyticum collagenase I and II, wherein saidmethod comprises testing a bacterial production strain for the absenceof a functional, secreted hemolytic toxin according to a method of theinvention.

In yet another embodiment, the invention is a method of purifying acrude collagenase composition, wherein said method comprises purifyingthe composition by filtration and column chromatography, followed byconfirming the absence of a hemolytic toxin according to a methoddescribed herein.

In a further embodiment, the invention is a method of treating acollagen-mediated condition in a patient in need thereof, wherein saidmethod comprises administering to said patient an effective amount of adrug product comprising collagenase, wherein the absence of a hemolytictoxin in said drug product or in a bacterial production strain producingsaid collagenase is confirmed according to a method of the inventionprior to administration of said drug product to a patient, and/orformulation of the collagenase in a pharmaceutical composition.

Kits for testing for the presence or absence of hemolytic toxins in asample are also described.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 shows protein alignment of Clostridium septicum (C. septicum)alpha toxin with the putative alpha toxin of C. histolyticum CLH_2834and 2835. The C. septicum alpha toxin amino acid sequence (SEQ ID NO: 7)is the upper sequence in each row. The C. histolyticum CLH_2834 & 2835(SEQ ID NO: 8) is the lower sequence in each row. The underlined, shadedsequence is the N-terminus of the mature C. septicum alpha toxin. Theasterisks above the amino acids shows non-conserved essential residuescritical for functionality (identifies mismatch in sequence). Theshading shows conserved essential residues (confirms identity). Thesequence numbering is based on the C. septicum sequence.

FIG. 2 shows blood agar plating of C. septicum. The arrows indicate betahemolytic activity.

FIG. 3 shows amino acid alignment of Bacillus proteolyticus thermolysinwith the putative delta toxin of C. histolyticum CLH_2576. The uppersequence in each row shows the sequence of Bacillus proteolyticus (B.proteolyticus) thermolysin protein (SEQ ID NO: 9). The lower sequence ineach row is the sequence of C. histolyticum CLH_2576 (SEQ ID NO: 10).The green shading shows the proprotein region. The numbering is based onthe thermolysin sequence.

FIG. 4 shows the prosequence amino acid alignment of B. proteolyticusthermolysin with the putative delta toxin of C. histolyticum CLH_2576.The upper sequence in each row is the prosequence of B. proteolyticusthermolysin protein (SEQ ID NO: 11). The lower sequence in each row isthe prosequence of C. histolyticum CLH_2576 (SEQ ID NO: 12). Theasterisks above the amino acids show the non-conserved essentialresidues critical for functionality (identifies mismatch in sequence).The green shading shows the conserved essential residues (confirmsidentity). The numbering is based on the thermolysin sequence.

FIG. 5 shows the mature sequence protein alignment of B. proteolyticusthermolysin with the putative delta toxin of C. histolyticum CLH_2576.The upper sequence in each row is the mature sequence of B.proteolyticus thermolysin (SEQ ID NO: 13). The lower sequence in eachrow is C. histolyticum CLH_2576 (SEQ ID NO: 14). The asterisks above theamino acids show non-conserved essential residues critical forfunctionality (identifies mismatch in sequence). The green shading showsconserved essential residues (confirms identity). The numbering is basedon thermolysin sequence.

FIG. 6 shows the protein alignment of C. perfringens perfringolysin withthe putative epsilon toxin of C. histolyticum CLH_1920. The uppersequence in each row is C. perfringens perfringolysin amino acidsequence (SEQ ID NO: 15). The lower sequence in each row is the aminoacid sequence of C. histolyticum CLH_1920 (SEQ ID NO: 16). The blue starshows the signal peptidase cleavage site of perfringolysin K43. Theasterisks above the amino acids show non-conserved essential residuescritical for functionality (identifies mismatch in sequence). The greenshading shows conserved essential residues (confirms identity). Thenumbering is based on the perfringolysin sequence.

FIG. 7 shows the beta hemolytic phenotype of tetanolysin.

FIG. 8 shows the protein alignment of C. histolyticum clostripain withthe putative gamma toxin of C. histolyticum CLH_1861. The upper sequencein each row is C. histolyticum clostripain amino acid sequence (SEQ IDNO: 17). The lower sequence in each row is C. histolyticum CLH_1920amino acid sequence (SEQ ID NO: 18). The asterisks above the amino acidsshows non-conserved essential residues critical for functionality(identifies mismatch in sequence). The green shading shows the conservedessential residues (confirms identity). The numbering based onclostripain X63673 sequence.

FIG. 9 shows an alignment comparison of the translated amino acidsequence from colG and the amino acid sequence of SEQ ID NO: 3 (thetranslated amino acid sequence from CLH_1768 and 1769; the uppersequence). As shown in FIG. 9, the mature protein encoded by the aminoacid sequence of SEQ ID NO: 3 differs from the translated amino acidsequence from colG amino acid sequence by three amino acids. TheN-terminus of the mature protein begins at Ile 119 of the sequence ofSEQ ID NO: 3. The amino acid sequence of the mature protein beginning atIle 119 of SEQ ID NO: 3 is SEQ ID NO: 5.

FIG. 10 shows an alignment comparison of the translated amino acidsequence from colH and SEQ ID NO: 4 (the translated amino acid sequencefrom CLH_2116; the bottom sequence). As shown in FIG. 10, the matureprotein encoded by the amino acid sequence of SEQ ID NO: 4 differs fromthe translated colG amino acid sequence by eight amino acids. TheN-terminus of the mature protein begins at Ala 31 in colG and in SEQ IDNO: 4. The amino acid sequence of the mature protein beginning at Ala 31of SEQ ID NO: 4 is SEQ ID NO: 6.

FIG. 11 shows the nucleotide sequence of SEQ ID NO: 1 (CLH_1768 and1769; collagenase I).

FIG. 12 shows the nucleotide sequence of SEQ ID NO: 2 (CLH_2116;collagenase II).

FIGS. 13A and 13B show the amino acid and nucleotide sequence of SEQ IDNO: 8 and SEQ ID NO: 21, respectively (CLH_2835 and CLH_2834; alphatoxin).

FIGS. 14A and 14B show the amino acid and nucleotide sequence of SEQ IDNO: 10 and SEQ ID NO: 22, respectively (CLH_2576; delta toxin).

FIGS. 15A and 15B show the amino acid and nucleotide sequence of SEQ IDNO: 16 and SEQ ID NO: 23, respectively (CLH_1920; epsilon toxin).

FIGS. 16A and 16B show the amino acid and nucleotide sequence of SEQ IDNO: 18 and SEQ ID NO: 24, respectively (CLH_1861; gamma toxin).

FIG. 17A shows the amino acid sequence of SEQ ID NO: 3 (colG).

FIG. 17B shows the amino acid sequence of SEQ ID NO: 5.

FIG. 18A shows the amino acid sequence of SEQ ID NO: 4 (colH).

FIG. 18B shows the amino acid sequence of SEQ ID NO: 6.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The words “a” or “an” are meant to encompass one or more, unlessotherwise specified. For example, “a hemoloytic toxin” refers to one ormore hemolytic toxins.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell culture, molecular biology,microbiology, cell biology, and immunology, which are well within theskill of the art. Such techniques are fully explained in the literature.See, e.g., Sambrook et al., 1989, “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press; Ausubel et al. (1995),“Short Protocols in Molecular Biology”, John Wiley and Sons; Methods inEnzymology (several volumes); Methods in Cell Biology (several volumes),and Methods in Molecular Biology (several volumes); the contents of eachof which are expressly incorporated by reference herein.

A. Recombinant Nucleic Acids and Proteins

A major source of collagenase is from the fermentation of C.histolyticum. An injectable formulation comprising C. histolyticumcollagenase I and collagenase II is sold under the trade name XIAFLEX®and is approved by the U.S. Food and Drug Administration for thetreatment of Dupuytren's contracture. Amino acid sequences forcollagenase I and collagenase II encoded by the colG and colH genes,respectively, have been described in the literature. For example, colGis described in GenBank Acc. No. D87215 and Matsushita et al. (1999),Journal of Bacteriology 181(3): 923-933, and colH has been described inGenBank Acc. No. D29981 and Yoshihara et al. (1994), Journal ofBacteriology 176(21): 6489-6496, the contents of each of which areexpressly incorporated by reference herein. The present invention isbased partially on sequencing analysis of the genes encoding collagenaseI and collagenase II in a C. histolyticum strain (Clone 004 describedbelow in the Examples) which produces and secretes functionalcollagenase I and collagenase II. The nucleotide sequences of the genesencoding collagenase I and collagenase II were found to be differentfrom the literature-described sequences for C. histolyticum (e.g.,GenBank Acc. Nos. D87125 and D29981) (SEQ ID NO: 19 and 20) (FIGS. 9 and10).

Collagenase I and collagenase II are metalloproteases and requiretightly bound zinc and loosely bound calcium for their activity (EddieL. Angleton and H. E. Van Wart, Biochemistry 1988, 27, 7406-7412).Collagenase I and collagenase II have broad specificity toward all typesof collagen (Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260 p2771-2′7′76). Collagenase I and collagenase II digest collagen byhydrolyzing the triple-helical region of collagen under physiologicalconditions (Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260 p2771-2776). Even though each collagenase shows different specificity(e.g., each has a different preferred amino sequence for cleavage),together, they have synergistic activity toward collagen (Mandl, I.,(1964), Biochemistry, 3: p. 1737-1′741; Vos-Scheperkeuter, G H, (1997),Cell Transplantation, 6: p. 403-412).

The invention encompasses a recombinant nucleic acid molecule comprisingor consisting of a polynucleotide of SEQ ID NO: 1 or the complement ofSEQ ID NO: 1. In certain aspects, the recombinant nucleic acid furthercomprises a heterologous regulatory sequence operably linked to thepolynucleotide. The invention further encompasses a recombinant nucleicacid molecule comprising or consisting of a polynucleotide of SEQ ID NO:2 or the complement of SEQ ID NO: 2. In certain aspects, the recombinantnucleic acid further comprises a heterologous promoter operativelylinked to the polynucleotide.

The invention also encompasses recombinant polypeptides encoded by therecombinant nucleic acids described herein. In some aspects, therecombinant polypeptides are encoded by the recombinant nucleic acidscomprising or consisting of a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments,the recombinant polypeptide comprises the amino acid sequence of SEQ IDNO: 3 or SEQ ID NO: 4. In additional embodiments, the recombinantpolypeptide comprises the amino acid sequence of SEQ ID NO:5 (the maturecollagenase I protein, beginning at Ile 119 of SEQ ID NO: 3 in FIG. 9)or SEQ ID NO:6 (the mature collagenase II protein, beginning at Ala 31of SEQ ID NO: 4 in FIG. 10). In yet another embodiment, the recombinantpolypeptide consists of the amino acid sequence of SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

In yet another embodiment, the invention is directed to a recombinantnucleic acid that encodes a polypeptide which comprises or consists ofthe amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In a furtherembodiment, the invention is directed to a recombinant nucleic acid thatencodes a polypeptide which comprises or consists of the amino acidsequence of SEQ ID NO: 5 or SEQ ID NO: 6. In a further aspect, therecombinant nucleic acid comprises a nucleotide sequence that encodes apolypeptide of amino acid sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5 or SEQ ID NO: 6.

A recombinant nucleic acid is a nucleic acid molecule that contains, inaddition to a polynucleotide sequence described herein (for example, thepolynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2), a furtherheterologous coding or non-coding nucleotide sequence. The term“heterologous” means that the polynucleotide originates from a differentspecies or from the same species, however, from another location in thegenome than said added nucleotide sequence. Recombinant polypeptides orproteins refer to polypeptides or proteins produced using recombinanttechniques, for example, those proteins or polypeptides produced fromcells transformed by an exogenous nucleic acid construct encoding thedesired polypeptide or protein.

The invention also relates to nucleic acids comprising thepolynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein saidnucleic acid is operatively linked to a regulatory sequence. Theinvention further relates to nucleic acids comprising a polynucleotidethat encodes a polypeptide comprising the amino acid sequence of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, wherein said nucleicacid is operatively linked to a regulatory sequence. Regulatorysequences include those regulatory sequences which direct constitutiveexpression of a nucleotide sequence in many types of host cells and/orthose which direct expression of the nucleotide sequence only in certainhost cells (e. g., tissue-specific regulatory sequences). Non-limitingexamples of regulatory sequences are promoters and enhancers. Regulatorysequences also include other expression control elements, for example,those described in Goeddei, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990), the contentsof which are expressly incorporated by reference herein. A nucleic acidis “operably linked” to a regulatory sequence when the nucleic acidmolecule is linked to the regulatory sequence in a manner which allowsexpression of the nucleic acid sequence.

A nucleic acid molecule described herein can additionally be fused to amarker sequence, for example, a sequence that encodes a polypeptide toassist in isolation or purification of the polypeptide. Such sequencesinclude, but are not limited to, those which encode aglutathione-S-transferase (GST) fusion protein, those which encode ahemaglutin A (HA) polypeptide marker from influenza, and those whichencode hexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.). In certain aspects, the invention is directed to apolypeptide comprising an amino acid sequence of SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein said polypeptide is fuseda marker amino acid sequence.

In a further aspect, the invention is directed to a nucleic acid that isa variant of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Avariant nucleic acid is a nucleic acid that includes an nucleotidesubstitution, addition or deletion relative to nucleotide sequence ofSEQ ID NO: 1 or SEQ ID NO: 2. In some aspects, the variant is a nucleicacid that encodes identical or substantially identical amino acidsequences as that of the nucleotide sequences of SEQ ID NO: 1 or SEQ IDNO: 2. As will be understood by the skilled artisan, because of thedegeneracy of the genetic code, several different nucleic acid sequencescan encode a given protein. For instance, the codons GCA, GCC, GCG andGCU each encode the amino acid alanine. Thus, for example, at everyposition where a specific amino acid is specified by one codon, thecodon can be changed to any of the corresponding codons that encode thesame amino acid without altering the amino acid sequence of the encodedpolypeptide. One of ordinary skill in the art will understand that eachcodon in a nucleotide sequence (except AUG, which is the only codon formethionine, and TGG, which is usually the only codon for tryptophan) canbe modified to yield a functionally identical molecule.

In certain embodiments, the invention is directed to polypeptidecomprising or consisting of amino acid sequence of SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, wherein one or more amino acidshave been deleted or added, wherein the polypeptide possesses theactivity of degrading or lysing collagen. In yet an additionalembodiment, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6,wherein one or more amino acid residues have been replaced with adifferent amino acid residue, wherein the polypeptide possesses theactivity of degrading or lysing collagen and wherein the polypeptidecomprises or consists of an amino acid sequence that is different fromthe amino acid sequences of GenBank Acc. Nos. D87125 (SEQ ID NO: 19) andD29981 (SEQ ID NO: 20). In certain aspects, when an amino acid isreplaced, the replacement is a conservative amino acid change. Aconservative amino acid change is, for example, substitution of anonpolar amino acid for another nonpolar amino acid or substitution of apolar amino acid for another polar amino acid or substitution of apositively charged amino acid for another positively charged amino acid,and the like. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid.

An isolated nucleic acid and an isolated polypeptide are not in the formor environment in which they exist in nature. For example, an isolatednucleic acid is one that is separated from the nucleotides whichnormally flanks the nucleic acid molecule in nature. Recombinant nucleicacids and recombinant nucleic acids within a vector are also an exampleof an isolated nucleic acid. Also, isolated nucleic acid moleculesinclude recombinant nucleic acid molecules in heterologous host cells,as well as partially or substantially purified nucleic acid molecules insolution.

As described in more detail below, the invention also encompassesrecombinant host cells, such as bacterial cells, fungal cells, plantscells, insect cells, avian cells, amphibian cells and mammalian cells,comprising the nucleic acid molecules described herein.

An expression cassette is a nucleotide sequence which is capable ofaffecting expression of a structural gene (i.e., a protein codingsequence, such as a collagenase of the invention) in a host compatiblewith such sequences. Expression cassettes can include a promoteroperably linked with the polypeptide coding sequence; and, optionally,with other sequences, e.g., transcription termination signals.Additional factors necessary or helpful in effecting expression may alsobe used, e.g., enhancers.

The invention also relates to vectors comprising a nucleic acid of theinvention. In one embodiment, the nucleic acid is SEQ ID NO: 1 or SEQ IDNO: 2, or a complement thereof. In another embodiment, the nucleic acidis a nucleic acid that encodes a polypeptide having the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. A“vector” is a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. A non-limiting example of avector is a plasmid which is a circular double stranded DNA into whichan additional DNA segment can be ligated. Another example of a vector isa viral vector, wherein an additional DNA segment is ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Expression vectors arecapable of directing the expression of genes to which they are operablylinked. Such expression vectors include, for example, plasmids. Theinvention encompasses other expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses) that are capable of directing gene expression.As will be appreciated by the skilled artisan, the design of theexpression vector depends on several factors, such as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. Expression vectors include one or more regulatorysequences which are selected based on the host cell to be used forexpression. As discussed above, the regulatory sequence is operablylinked to the nucleic acid to be expressed, for example, a nucleic acidof the invention. In some embodiments, the regulatory sequence is aregulatory sequence native to the transformed host cell. An expressionvector can comprise one or more selectable markers, including, but notlimited to, the gene that encodes dihydrofolate reductase and the genesthat confer resistance to neomycin, tetracycline, ampicillin,chloramphenicol, kanamycin and streptomycin resistance.

Prokaryotic and eukaryotic host cells can be transfected by the vectorsdescribed herein. Host cells which can be transfected with the vectorsof the present invention include, but are not limited to, bacterialcells such as E. coli (e.g., E. coli K12 strains), Streptomyces,Pseudomonas, Serratia marcescens and Salmonella typhimurium, insectcells (baculovirus), including Drosophila, fungal cells, such as yeastcells, plant cells and mammalian cells, such as thymocytes, Chinesehamster ovary cells (CHO), COS cells, and Lactococcus lactis cells. Insome embodiments, the host cell is a bacterial cell. In yet anotherembodiment, the host cell is an E. coli strain. In yet an additionalembodiment, the host cell is Lactococcus lactis cell. Methods for theproduction of recombinant polypeptides in Lactococcus lactis bacteriahave been described, for example, in U.S. Pat. No. 7,358,067, thecontents of which are expressly incorporated by reference herein. In oneembodiment, the host cell is Lactococcus lactis and the nucleic acidcomprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2operably linked to pH regulatable promoter P170 and derivatives thereof.The P170 promoter and derivatives thereof have been described in detailin WO 94/16086 and WO 98/10079, the contents of which are incorporatedby reference herein.

Ligating the nucleic acid molecule into a gene construct, such as anexpression vector, and transforming or transfecting into hosts, eithereukaryotic (yeast, avian, insect, plant or mammalian) or prokaryotic(bacterial cells), are standard procedures. A vector described hereincan be introduced into prokaryotic or eukaryotic cells usingconventional transformation or transfection techniques, including, butnot limited to, calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, or electroporation. Thepolypeptides of the present invention can be isolated or purified (e.g.,to homogeneity) from recombinant cell culture by a variety of processes.

The invention encompasses methods of producing a functional collagenaseI or collagenase II or a combination thereof comprising culturing a hostcell transformed or transfected with a vector comprising a nucleic acidof the invention. The method additionally comprises isolating thepolypeptide from the medium or the host cell. A functional collagenaseis a polypeptide that has a biological activity of a naturally-occurringcollagenase, for example, a collagenase that possesses the ability todegrade collagen.

The polypeptide can be isolated by methods including, but not limitedto, anion or cation exchange chromatography, ethanol precipitation,affinity chromatography and high performance liquid chromatography(HPLC), or a combination of any of thereof. The particular method usedwill depend upon the properties of the polypeptide and the selection ofthe host cell; appropriate methods will be readily apparent to thoseskilled in the art.

In some embodiments, the invention is a method of producing collagenaseI or collagenase II, said method comprising the steps of (i)constructing a recombinant bacterium comprising the nucleotide sequenceof SEQ ID NO: 1 or SEQ ID NO:2, or the polynucleotide encoding thepolypeptide sequence of SEQ ID NO: 3, 4, 5 or 6 operably linked to anappropriate regulatory sequence; (ii) cultivating said recombinantbacterium under suitable conditions to express the gene, and (iii)harvesting from the recombinant bacterium, the collagenase I orcollagenase II. The collagenase I and collagenase II can be purified bya variety of methods known to those skilled in the art, including dyeligand affinity chromatography, heparin affinity chromatography,ammonium sulfate precipitation, hydroxylapatite chromatography, sizeexclusion chromatography, ion exchange chromatography, and metalchelation chromatography. In some embodiments, the collagenase I andcollagenase II are purified via filtration and column chromatography andthe purified collagenase I and II are combined in a ratio of about 1 to1 using methods described in U.S. Pat. No. 7,811,250, the contents ofwhich are expressly incorporated by reference herein.

Examples of collagen mediated-diseases that can be treated by thecompositions (comprising collagenase I, collagenase II, or a combinationthereof encoded by the nucleic acids described herein and/or comprisingthe amino acid sequences of SEQ ID NO: 3 and/or SEQ ID NO: 4) andmethods of the invention include, but are not limited to, Dupuytren'sdisease, Peyronie's disease, frozen shoulder (adhesive capsulitis),keloids, hypertrophic scars, depressed scars, such as those resultingfrom inflammatory acne; post-surgical adhesions, acne vulgaris, lipomas,and disfiguring conditions such as wrinkling, cellulite formation andneoplastic fibrosis. U.S. Pat. Nos. 6,086,872 and 5,589,171,incorporated herein by reference, disclose the use of collagenasepreparations in the treatment of Dupuytren's disease. U.S. Pat. No.6,022,539, incorporated herein by reference, discloses the use ofcollagenase preparations in the treatment of Peyronie's disease.

In addition to its use in treating collagen-mediated diseases, acomposition comprising a recombinant polypeptide described herein isalso useful for the dissociation of tissue into individual cells andcell clusters as is useful in a wide variety of laboratory, diagnosticand therapeutic applications. These applications involve the isolationof many types of cells for various uses, including microvascularendothelial cells for small diameter synthetic vascular graft seeding,hepatocytes for gene therapy, drug toxicology screening andextracorporeal liver assist devices, chondrocytes for cartilageregeneration, and islets of Langerhans for the treatment ofinsulin-dependent diabetes mellitus. Enzyme treatment works to fragmentextracellular matrix proteins and proteins which maintain cell-to-cellcontact. Since collagen is the principle protein component of tissueultrastructure, the enzyme collagenase has been frequently used toaccomplish the desired tissue disintegration. In general, thecomposition of the present invention is useful for any application wherethe removal of cells or the modification of an extracellular matrix, aredesired.

The invention encompasses pharmaceutical compositions comprising apharmaceutically acceptable carrier and collagenase I and/or collagenaseII produced according to a method described herein. In yet anotherembodiment, the pharmaceutical compositions comprises collagenase Icomprising or consisting of the amino acid sequence of SEQ ID NO: 3 orSEQ ID NO: 5. In a further embodiment, the pharmaceutical compositioncomprises collagenase II comprising or consisting of the amino acidsequence of SEQ ID NO: 4 or SEQ ID NO: 6. In yet another aspect, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier and a collagenase I and collagenase II as described herein. In afurther aspect, the pharmaceutical composition comprises apharmaceutically acceptable carrier and the collagenase I andcollagenase II at 1:1 mass ratio. The pharmaceutical composition of thepresent invention comprises an effective amount of a collagenase thepresent invention formulated together with one or more pharmaceuticallyacceptable carriers or excipients.

B. Methods of Detecting the Presence of a Hemolytic Toxin

In some embodiments, the invention encompasses methods of detecting thepresence of a hemolytic toxin in a bacterial fermentation, wherein thebacterial fermentation produces a collagenase. In certain aspects, theinvention provides a method for releasing a collagenase drug productprior to the therapeutic administration of said collagenase drugsubstance to a patient comprising detecting the presence of a hemolytictoxin in the drug product production strain. The term “drug productproduction strain,” “production strain,” “collagenase productionstrain,” and “bacterial production strain” are used interchangeably andrefer to a bacterial strain from which a collagenase is obtained. Inother aspects, the invention provides a method for releasing acollagenase drug product prior to the therapeutic administration of saidcollagenase drug product to a patient, comprising detecting the presenceof a hemolytic toxin in the drug product.

As used herein, the phrase “releasing a collagenase drug product” meansto confirm the absence of a hemolytic toxin in the collagenase drugproduct. It is understood that the terms “drug substance”, “drugproduct” or “collagenase composition” can be used interchangeably. Alsoas used herein, the terms “hemolysin” and “hemolytic toxin” are usedinterchangeably, and refer to a toxin that is responsible for the lysisof a red blood cell.

It has been discovered that the collagenase production strain and drugproduct can be assayed for the presence or absence of hemolyticactivity, ensuring that the collagenase drug substance provides a highlyreproducible and optimal enzymatic activity and superior therapeuticeffect, while lowering the potential for side effects. In accordancewith the invention, methods are provided for assaying the productionstrain or drug product for the secretion or presence of a functionalhemolytic toxin that may be co-present with collagenase in the drugproduct. The invention encompasses a method of assaying a test samplefor the presence of a hemolytic toxin, wherein the test sample comprisesa bacterial production strain or a collagenase composition, comprisingincubating the test sample with red blood cells, followed by detectionof lysis of red blood cells.

Specific methods for detecting lysis of red blood cells are describedthroughout the literature, including, for example, 1) Ryan K J and Ray CG. Principles of laboratory diagnosis. In Sherris medical microbiology:an introduction to infectious diseases. Ryan K J, Ray C G, and Sherris JC (eds.) McGraw-Hill Professional, 2004; 229-260; and 2) Eschbach E etal. Improved erythrocyte lysis assay in microtitre plates for sensitivedetection and efficient measurement of hemolytic compounds fromichthyotoxic algae. Journal of Applied Toxicology 21, 513-519 (2001),the contents of each of which are expressly incorporated by referenceherein.

In one embodiment of the invention, the method comprises incubatingsamples of a collagenase production strain, a partially purifiedcollagenase isolated from a collagenase production strain, or acollagenase drug product on a blood agar substrate, and observing theblood agar for zones of clearance after the period of incubation,wherein a zone of clearance indicates the lysis of red blood cells. Ifthe bacterial product strain was tested, the lysis of red blood cellsindicates the secretion of a functional hemolytic toxin from thebacterial production strain. If a partially purified collagenase or acollagenase drug product was tested, the lysis of red blood cellsindicates the presence of a functional hemolytic toxin in the partiallypurified collagenase or in collagenase drug product. In certainembodiments, the production strain is a strain of C. histolyticum. Theabsence of a zone of clearance indicates the absence of a hemolytictoxin. The observed absence of zones of clearance indicate or confirmthe absence of hemolytic toxins in the collagenase production strain, inthe partially purified collagenase, or in the collagenase drug product,and allow the drug product to be released for therapeuticadministration.

In another embodiment, the method comprises incubating red blood cellswith extracts taken from a collagenase production strain, or with apartially purified collagenase isolated from a collagenase productionstrain, or with a collagenase drug product, followed by photometricallyanalyzing the incubation mixture for the lysis of red cells as indicatedby the appearance of hemoglobin in the incubation mixture. A hemolytictoxin will lyse the red blood cells, releasing hemoglobin into theincubated sample. The photometric detection of hemoglobin can provide asensitive assay for the presence of hemolytic toxins. In one aspect, redblood cells are incubated with extracts taken from a collagenaseproduction strain, or with a partially purified collagenase isolatedfrom a collagenase production strain, or with a collagenase drugproduct, and then photometrically analyzing the extracts for thepresence of hemoglobin at a wavelength of 540 nm. In another aspect, thephotometric analysis is performed at a wavelength of 414 nm. In yetanother aspect, incubation and photometric analysis can be performedusing microtiter plates. The absence of hemoglobin, and thus the absenceof hemolytic toxins, would allow the release of the drug product fortherapeutic administration to a patient.

Hemolytic toxins as found in C. histolyticum belong to two differentfamilies of hemolysins: aerolysin-like hemolysins, and oxygen-labilehemolysins. The aerolysin-like hemolysins are synthesized by thebacterium as inactive preproteins that are secreted into theextracellular environment as inactive protoxins. The inactive protoxinswill bind to receptors on a target cell membrane, for example, receptorson a red blood cell where the protoxins are cleaved into their activestructures by proteases. Once activated, the toxins oligomerize on thecell surface into a prepore complex, followed by insertion of abeta-barrel into the target cell membrane. The beta-barrel forms a porein the membrane, allowing the rapid influx of calcium ions into thecell, with toxic consequences to the cell. The alpha toxin of C.histolyticum is most likely an aerolysin-like hemolysin, as it has beendiscovered to share significant homology with Clostridium septicum alphatoxin, which is a member of the aerolysin-like family of toxins, andwhich possess hemolytic activity (see, for example, Example 1 below).

Epsilon toxin of C. histolyticum, and tetanolysin of Clostridium tetani(C. tetani), have been described as an oxygen-labile hemolysins[Hatheway C L. Clin Microbiol Rev 3(1): 66-98 (1990)]. Epsilon toxin ofC. histolyticum has been discovered to share homology with tetanloysin,which is a member of thiol-activated, beta-barrel, pore-forming toxinswith affinity for cholesterol. Such proteins are part of a family ofCholesterol Dependent Cytolysins (CDC). These proteins are secreted bythe bacterium into the extracellular environment as water-solublemonomeric proteins where they bind to target cell membranes, mediated bycholesterol binding. The toxin then oligomerizes on the membrane surfaceto form arcs and ring-like structures that are responsible forcytolysis. The epsilon toxin of C. histolyticum is known to be anoxygen-labile hemolysin, and is similar serologically to thoseoxygen-labile hemolysins produced by other strains of Clostridium, suchas C. tetani, C. novyi, and C. septicum.

In certain aspects, the invention is directed to a method of detectingthe presence of C. histolyticum alpha toxin in a bacterial productionstrain using an assay described herein. In other aspects, the inventionis directed to a method of detecting the presence of C. histolyticumalpha toxin in a drug product. In a further aspect, the invention isdirected to a method of detecting the presence of C. histolyticumepsilon toxin in a bacterial production strain. In yet another aspect,the invention is directed to a method of detecting the presence of C.histolyticum epsilon toxin in a drug product. In a still further aspect,the invention is directed to a method of detecting the presence of C.histolyticum alpha toxin and epsilon toxin in a bacterial productionstrain. In an additional embodiment, the invention is directed to amethod of detecting the presence of C. histolyticum alpha toxin andepsilon toxin in a drug product.

The invention also encompasses a method of producing a drug productconsisting of collagenase I and collagenase II, wherein the collagenaseI and II are obtained from C. histolyticum, and wherein the methodcomprises the steps of fermenting a strain of C. histolyticum in whichthe absence of a functional, secreted hemolytic toxin has been confirmedby incubating the production strain with red blood cells underconditions suitable for lysis of red blood cells by a hemolytic toxin,wherein lysis of red blood cells indicates secretion of a hemolytictoxin and wherein the absence of lysis of the red blood cells indicatesthe absence of a hemolytic toxin. In another aspect, the invention isdirected to a method of producing a drug product consisting ofcollagenase I and collagenase II, wherein the collagenase I and II areobtained from C. histolyticum, and wherein the method comprises thesteps of confirming the absence of a functional, secreted hemolytictoxin in the drug product by incubating the drug product with red bloodcells under conditions suitable for lysis of red blood cells by ahemolytic toxin, wherein lysis of red blood cells indicates secretion ofa hemolytic toxin and wherein the absence of lysis of the red bloodcells indicates the absence of a hemolytic toxin.

Further aspects of the invention include methods of purifying a crudecollagenase composition comprising purifying the composition byfiltration and column chromatography followed by confirming the absenceof a hemolytic toxin by incubating a sample of the purified compositionwith red blood cells under conditions suitable for lysis of red bloodcells by a hemolytic toxin, wherein lysis of red blood cells indicatessecretion of a hemolytic toxin and wherein the absence of lysis of thered blood cells indicates the absence of a hemolytic toxin.

As discussed above, several diseases and conditions are associated withexcess collagen deposition and the erratic accumulation of fibroustissue rich in collagen and can be treated with collagen drug products.Such diseases and conditions are collectively referred to herein as“collagen-mediated diseases”. The invention also encompasses a method oftreating a collagen-mediated disease in a patient in need thereof,wherein the composition comprising collagenase is administered to saidpatient and wherein, prior to said administration, said composition orbacterial production strain is assayed for the presence or absence ofhemolytic toxins using a method described herein. Examples of collagenmediated-conditions that may be treated by the compositions and methodsdescribed herein include but are not limited to: Dupuytren's disease;Peyronie's disease; frozen shoulder (adhesive capsulitis), keloids;hypertrophic scars; depressed scars such as those resulting frominflammatory acne; post-surgical adhesions; acne vulgaris; lipomas, anddisfiguring conditions such as wrinkling, cellulite formation andneoplastic fibrosis. In certain aspects, the assayed composition isadministered to a patient to treat Peyronie's or Duputyren's diseases oradhesive capsulitis.

With respect to the production strain that can be assayed according to amethod of the invention, it is known, for example, that collagenase isexpressed by bacteria that are members of the genera Actinobacillus,Actinomadura, Bacillus, Bacteroides, Bifidobacterium, Brucella,Capnocytophaga, Clostridium, Enterococcus, Escherichia, Eubacterium,Flavobacterium, Fusobacterium, Peptococcus, Peptostreptococcus,Porphyromonas, Prevotella, Proteus, Pseudomonas, Serratia,Staphylococcus, Streptomyces, Streptococcus, Treponema, and Vibrio. Inone embodiment of the invention, the production strain is selected fromthe above listed genera. In another embodiment, the production strain isan E. coli strain, including forms of E. coli that have been transformedwith recombinant forms of collagenase I and collagenase II. In a morepreferred embodiment, the production strain is a C. perfringens strain.In a most preferred embodiment, the production strain is a C.histolyticum (C. his) strain.

In certain aspects, the production strain produces a collagenasecomposition comprising a mixture of collagenase I and collagenase II. Ina further embodiment, the production strain used to produce a mixture ofcollagenase I and collagenase I is C. histolyticum. In anotherembodiment, the collagenase drug product comprises a mixture of highlypurified C. histolyticum collagenase I and collagenase II in a massratio of about 1 to 1.

Kits for testing for the presence of hemolysins in a sample are alsopresented, wherein a hemolysin is a substance that causes lysis of redblood cells. The kits allow the identification of test substances thatare hemolytic, or contain, hemolysins. Test substances include, but arenot limited to, chemical, biological, and radiation-emitting substances.In one embodiment, the kit comprises materials for testing for thepresence of hemolysins in a test sample including, for example, a kitcomprising red blood cells and related test materials. In anotherembodiment, the kit comprises a petri dish comprised of blood agar, apositive control, and a negative control comprised of a bacterial strainwherein the hemolytic genes are mutated or knocked out, and wherein nofunctional hemolytic proteins are produced. In yet another embodiment,the kit comprises red blood cells, microtiter plates, a positivecontrol, and a negative control comprised of the drug product.

As will be understood, the inventive kits and methods can be used todetect the presence or absence of hemolysins in collagenasecompositions, wherein the collagenase is obtained from a bacteria.

The crude collagenase obtained from C. histolyticum can be purified by avariety of methods known to those skilled in the art, including dyeligand affinity chromatography, heparin affinity chromatography,ammonium sulfate precipitation, hydroxylapatite chromatography, sizeexclusion chromatography, ion exchange chromatography, and metalchelation chromatography. Crude and partially purified collagenase iscommercially available from many sources including Advance BiofacturesCorp., Lynbrook, N.Y. Methods of purification of crude collagenaseobtained from C. histolyticum are also described in U.S. Pat. No.7,811,560, the contents of which are expressly incorporated herein byreference. In certain embodiments, the purification procedure comprisesthe steps of: a) filtering the crude harvest through a MUSTANG Qanion-exchange capsule filter; b) adding ammonium sulphate; preferablyto a final concentration of 1M; c) filtering the crude harvest;preferably through a 0.45 μm filter; d) subjecting the filtrate througha HIC column; preferably a phenyl sepharose 6FF (low sub); e) addingleupeptin to the filtrate; preferably to a final concentration of 0.2 μmto post HIC eluted product; f) removing the ammonium sulfate andmaintaining leupeptin for correct binding of collagenase I andcollagenase II with buffer exchange by TFF; preferably with bufferexchange by TFF; g) filtering the mixture of step; (f) preferablythrough a 0.45 μm filter; h) separating collagenase I and collagenase IIusing Q-Sepharose HP; i) preparing TFF concentration and formulation forcollagenase I and collagenase II separately; wherein TFF is a tangentialflow filtration using 10 and/or 30 K MWCO (molecular weight cut-off) PESor RC-polyethersulfone or regenerated cellulose filter membranes (TFFprovides a means to retain and concentrate select protein and exchangethe protein from one buffer solution into another); and j) filteringthrough a 0.2 μm filtration system.

C. C. histolyticum Alpha, Beta, Delta, Epsilon and Gamma Toxins

The amino acid sequences of the alpha, delta and epsilon toxins of C.histolyticum Clone 004 are shown in the Figures and are SEQ ID NO: 8,SEQ ID NO: 12 and SEQ ID NO: 16, respectively. The nucleotide sequencesof the alpha, delta and epsilon toxins of C. histolyticum Clone 004 arealso shown in the Figures and are SEQ ID NO: 21, SEQ ID NO: 22, and SEQID NO: 23, respectively. Each of the amino acid sequences of SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO: 16 have sequence characteristics thatrender these proteins non-functional and/or unsecreted.

For the gamma toxin (clostripain), there are only three amino aciddifferences when compared to the model protein (see Examples section)and none of the amino acid residues which are found to differ in the C.histolyticum Clone 004 gamma toxin have been identified as essential foractivity. Thus, it is predicted that the C. histolyticum Clone 004 gammatoxin (having the amino acid sequence of SEQ ID NO: 18) is secreted andfunctional. The nucleotide sequence of the C. histolyticum Clone 004gamma toxin is SEQ ID NO: 24.

As discussed above, the beta toxins having amino acid sequences of SEQID NO: 3 and SEQ ID NO: 4 are fully functional.

As will be understood, one or more mutations (for example, deletion oraddition of one or more amino acid residues or nucleic acid residues)can be introduced into the nucleotide and/or amino acid sequences of C.histolyticum alpha, beta, epsilon or gamma toxins (SEQ ID NO: 8, SEQ IDNO: 12, SEQ ID NO: 16 and SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23 and SEQ ID NO: 24). In certain aspects, one or mutationsare introduced in order to improve or impair the activity, function,production and/or secretion of the toxin. In one embodiment, a mutationcan be introduced that renders the alpha, beta, and/or epsilon toxinsfunctional and/or secreted. In another embodiment, the sequence of thegamma toxin (SEQ ID NO: 18) can be mutated so as to render the proteinnon-functional and/or unsecreted.

Also encompassed by the present invention are methods of producingantibodies against C. histolyticum or a C. histolyticum toxin comprisingadministering to a subject an effective amount of a compositioncomprising a protein or peptide, wherein said protein or peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ IDNO: 6, or a fragment or variant thereof, or a combination of any ofthereof. In addition, the present invention includes methods ofstimulating an immune response to a C. histolyticum toxin comprisingadministering to a subject an effective amount of a compositioncomprising a protein or peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or a fragment or variantthereof, or a combination of any of thereof. The invention also includesa vaccine comprising an effective amount of a protein or peptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ IDNO: 6, or a fragment or variant thereof, or a combination of any ofthereof. The protein or peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or a fragment or variantthereof can be produced by a C. histolyticum strain or can be arecombinant protein or peptide.

D. Pharmaceutical Compositions Comprising Collagenase and Methods ofTreatment

The invention described herein encompass pharmaceutical compositionscomprising the protein sequences and recombinant proteins and also,pharmaceutical compositions comprising a collagenase drug productassayed according to methods described herein. As used herein, the term“pharmaceutically acceptable carrier or excipient” means a non-toxic,inert solid, semi-solid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. “Treating” or “treatment”includes the administration of the compositions, compounds or agents ofaspects of the present invention to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease,alleviating or ameliorating the symptoms or arresting or inhibitingfurther development of the disease, condition, or disorder. A“therapeutically effective amount” or an “effective amount” is an amountwhich, alone or in combination with one or more other active agents, cancontrol, decrease, inhibit, ameliorate, prevent or otherwise affect oneor more symptoms of a disease or condition to be treated. In the contextof producing an immune response or in the preparation of a vaccine, an“effective amount” encompasses an amount effective to produce an immuneresponse, including the generation of antibodies against an antigen.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; glycolssuch as propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, perfuming agents, preservatives and antioxidants canalso be present in the composition, according to the judgment of theformulator.

Collagenase compositions can also be prepared by mixing either aspecific number of activity units or specific masses of the preferablypurified enzymes. Collagenase activity can be measured by the enzyme'sability to hydrolyze either synthetic peptide or collagen substrate.Those skilled in the art will recognize that enzyme assays other thanthose disclosed herein may also be used to define and preparefunctionally equivalent enzyme compositions. Collagenase activity can bedescribed, for example, in SRC units. One SRC unit will solubilize rattail collagen into ninhydrin reaction material equivalent to 1 nanomoleof leucine per minute, at 25° C. and pH 7.4. In certain embodiments ofthe present invention, collagenase activity is described in ABC units.This potency assay of collagenase is based on the digestion ofundenatured collagen (from bovine tendon) at pH 7.2 and 37° C. for 20-24hours. The number of peptide bonds cleaved is measured by reaction withninhydrin. Amino groups released by a trypsin digestion control aresubtracted. One net ABC unit of collagenase will solubilize ninhydrinreactive material equivalent to 1.09 nanomoles of leucine per minute. 1SRC unit equals approximately 6.3 ABC units.

In certain aspects, the drug substance for injectable collagenaseconsists of two microbial collagenases, referred to as Collagenase AUX Iand Collagenase ABC I and Collagenase AUX II and Collagenase ABC II. Itis understood that the terms “Collagenase I”, “ABC I”, “AUX I”,“collagenase AUX I”, and “collagenase ABC I” mean the same and can beused interchangeably. Similarly, the terms “Collagenase II”, “ABC II”,“AUX II”, “collagenase AUX II”, and “collagenase ABC II” refer to thesame enzyme and can also be used interchangeably. These collagenases aresecreted by bacterial cells. They are isolated and purified from C.histolyticum culture supernatant by chromatographic methods. Bothcollagenases are special proteases and share the same EC number (E.C.3.4.24.3).

Collagenase AUX I has a single polypeptide chain consisting ofapproximately 1000 amino acids with a molecular weight of 115 kDa.Collagenase AUX II has also a single polypeptide chain consisting ofabout 1000 amino acids with a molecular weight of 110 kDa.

In some embodiments, the drug substance (collagenase concentrate) has anapproximately 1 to 1 mass ratio for collagenase AUX I and AUX II. In oneembodiment, the collagenase concentrate has an extinction coefficient of1.528.

The pharmaceutical compositions of this invention can be administeredparenterally, topically, or via an implanted reservoir. The term“parenteral,” as used herein, includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. In a preferred embodiment, thecomposition is injected into the affected tissue. In the case ofPeyronie's or Duputyren's diseases or adhesive capsulitis, thecomposition is injected into the cord of the hand or the Peyronies'plaque. The term “local administration” is defined herein to embracesuch direct injection into the affected tissue. In certain aspects, thepharmaceutical composition of the invention is an injectableformulation. In certain additional aspects, the pharmaceuticalcomposition is a topical formulation.

Furthermore, depending on the treatment, improved results can, in somecircumstances, be obtained by immobilizing the site of injection afteradministration of the pharmaceutical composition. For example, the siteof administration (e.g., the hand), can be immobilized for 4 or morehours.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids, such as oleic acid, are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use. The sterile solutions may also be lyophilized forlater use.

In some embodiments, the composition comprising collagenase is alyophilized, injectable composition formulated with sucrose, Tris at apH level of about 8.0. Generally, a source of calcium is included in theformulation, such as calcium chloride.

Dosage forms for topical or transdermal administration of apharmaceutical compositions of this invention include ointments, pastes,creams, lotions, gels, powders, solutions, sprays, inhalants or patches.The active component is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as may be required.

The ointments, pastes, creams and gels may contain, in addition to apolypeptide of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to the polypeptides of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of an active agent to the body. Such dosage forms can be madeby dissolving or dispensing the active agent in the proper medium.Absorption enhancers can also be used to increase the flux of thepolypeptide across the skin. The rate can be controlled by eitherproviding a rate-controlling membrane or by dispersing the polypeptideof the invention in a polymer matrix or gel.

Therapeutic administration of the pharmaceutical may be parenterally,topically, or via an implanted reservoir. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques. The term “local administration” is defined hereinto embrace such direct injection. In one embodiment, therapeuticadministration of the pharmaceutical composition is by injection.

Therapeutic administration of the pharmaceutical in dosage forms fortopical or transdermal administration include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants or patches. Theactive component is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of the drug product, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of the drugproduct, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

The invention will be better understood in connection with the followingexamples, which are intended as an illustration only and not limiting ofthe scope of the invention. Various changes and modifications to thedisclosed embodiments will be apparent to those skilled in the art andsuch changes and modifications may be made without departing from thespirit of the invention and the scope of the appended claims.

EXAMPLES Example 1: C. histolyticum Genome Sequencing and Toxin SequenceAnalysis

A dearth of scientific studies related to the C. histolyticum alpha,delta and epsilon (α, δ, and ε toxins) has resulted in limited knowledgeabout the protein structure of these toxins. To address this knowledgedeficit, a genome sequencing initiative was undertaken to more fullyunderstand the production organism with particular focus on theidentification of putative toxin genes. As a consequence of this effort,the complete genome of the Collagenase Clostridium Histolyticumproduction strain (Clone 004) (Auxilium Product Operation, Malvern, Pa.)has only recently been generated, representing apparently, the firsttime that the genome sequence of any C. histolyticum strain has beenreported.

There were three fundamental steps involved in the genome sequenceproject. First, genomic DNA was extracted from a Clone 004 cultivationand forwarded to Creative Genomics for sequencing (Shirley, N.Y., USA).The genome sequence of C. histolyticum Clone 004 was obtained usingindustry standard methods. Second, the results obtained from the genomesequence were analyzed using standard bioinformatics methods (BLASTanalysis) in order to query the sequence information against genomesequence databases. This second stage resulted in the assignment ofprotein information for each C. histolyticum gene that was identified.The use of two databases ensured a comprehensive evaluation but alsoserved as a second source to verify the protein assignment. The thirdstep in the project was a comparative analysis of the C. histolyticumputative toxin sequence with the protein assigned automatically by theBLAST analysis.

i. C. histolyticum Genome Sequencing and Identification of ModelProteins

Samples of genomic DNA isolated from an expansion of C. histolyticum(CLH) WCB derived from Clone 004 was forwarded to Creative Genomics(Shirley, N.Y., USA) for genome sequencing. Creative Genomics employedstandard methods used for sequence determination of genomic DNA samplessubmitted by clients. The genome sequence was generated from Roche/454GS-FLX system with titanium chemistry (fragment sequencing) accompaniedwith Illumina/Solexa Genome Analyzer. The ANI 3730x1 was employed toaccomplish genome finishing by primer walking. The entire genomesequence of 2,842,906 base pairs with a 29.44% GC content was completedand these values were typical of the genome size and GC content obtainedfor other Clostridial genomes. Each of the 2,887 open reading frames(ORFs) identified was assigned a unique CLH number. Each of the putative2,887 genes was further investigated using BLAST analysis of the GenBankand SwissProt databases resulted in the tentative assignment of the locifor beta, gamma, alpha, and epsilon toxins. The results of the initialassessment are presented in Table 1. Thus, the assignment of modelproteins was completed as a result of an automated analysis via acomprehensive search of two databases. The model protein assignment wasnot influenced by operator interpretation.

TABLE 1 Assignments of Model Proteins for Putative CLH Toxins based uponComparison with Two Sequence Databases Common Toxin CLH Name Name ModelProtein alpha CLH -2834 & 2835 Lethal factor Aerolysin/Hemolysin (C.septicum alpha toxin) beta CLH_1768 & 1769 Collagenase I Collagenase Ifrom colG beta CLH_2116 Collagenase II Collagenase II from colH epsilonCLH_1920 Oxygen labile C. perfringens hemolysin perfringolysin C. tetanitetanolysin gamma CLH_1861 Clostripain C. histolyticum clostripain

An inspection of the BLAST analysis results of the C. histolyticumgenome did not reveal an ORF coding for an elastase. However, proteaseshave been classified by MEROPS (MEROPS.sanger.ac.uk/) based upon thecriterion of the most prominent functional group in the active site ofthose proteases. Using this MEROPS based functional approach, anelastase falls into the M4 peptidase family of which thermolysin (EC3.4.24.27) is the best studied member of the family and is the classicalmodel for such proteases. Using this knowledge, a re-inspection of theBLAST analysis output suggested that C. histolyticum possesses a singleORF that shares significant homology with thermolysin. Therefore, theputative delta toxin gene within C. histolyticum has been assigned as ahomolog of B. proteolyticus thermolysin.

The results of the initial assessment are presented in Table 2 belowbased upon comparison with two sequence databases.

TABLE 2 Assignments of Model Proteins for Putative CLH Toxins CommonToxin CLH Name Name Model Protein alpha CLH 2834 & 2835 Lethal factor C.septicum alpha toxin beta CLH_1768 & 1769 Collagenase I Collagenase Ifrom colG beta CLH_2116 Collagenase II Collagenase II from colH deltaCLH_2576 Elastase B. thermoproteolyticus thermolysin epsilon CLH_1920Oxygen labile C. perfringens hemolysin perfringolysin gamma CLH_1861Clostripain C. histolyticum clostripain

ii. Protein Sequence Alignments and Analysis

To identify the signal peptide, the sample and control sequences wereanalyzed in a program termed SignalP identify potential signal peptidesequences (Nielsen (2004), J. Glasgow et al., eds., Proc. Sixth Int.Conf. on Intelligent Systems for Molecular Biology, 122-130. AAAI Press,1998). A signal peptide is usually located within the first seventyamino acids (or the N-terminus region) of the protein sequence and actsas a signal sequence for the enzyme to be secreted. The signal peptideis cleaved and the resulting protein sequence is the mature protein.Using SignalP, the user can identify the signal peptide cleavage sitelocation in order to identify the N-terminus of the mature protein. Forsome sample sequences, in particular alpha toxin and beta toxin (AUX-I),however, only the mature protein was identified, not the entire proteinsequence including the signal peptide sequence. Further examinationrevealed that the sequence fragmentation procedure employed separatedthe signal peptide sequence from the mature protein. The mature proteinand signal sequences were reassembled and processed through thealignment tool.

Once all the protein sequences were collected, pair wise sequencealignments were constructed using MATLAB 7.0.10 (The MathWorks, Inc.,2010). Pair wise sequence alignments are direct comparisons of twosequences to determine the similarities and differences between twosequences. Both control and sample sequences were uploaded into MATLABand an alignment was made using the Needleman-Wunsch algorithm andBLOSUM50 scoring matrix. The algorithm and scoring matrix assist inassembling the alignment as the algorithm dictates the value of eachamino acid match or mismatch based off of the scoring matrix andincorporates gap values when necessary. Gaps can occur for multiplereasons, including, but not limited to, two sequences having varyinglengths and to ensure that the appropriate amino acids are matching upto one another. The scoring matrix is based off of substitution ratesobserved frequently among sequences and serves to rate the similarity ordissimilarity between two sequences (National Center for BiotechnologyInformation).

The Hatheway (1990) review (Clin Microbiol Rev 3: 66-98) indicated thatall five toxins were secreted proteins (exosubstances) and all fivetoxins had identifiable functionality. This information was used toconduct analysis of the putative CLH toxins. To analyze the proteinfunction of the putative CLH toxins, a number of model proteins wereselected based upon literature findings and BLAST results. The controlswere downloaded from the National Center for Biotechnology Information(NCBI) in Fasta format.

1. Alpha (α) Toxin

Sparse information related to C. histolyticum alpha toxin following thework of Bowen (1952) (Yale J Biol Med 25:124-138) exists in theliterature. Thus, the interrogation of the genome sequence for putativetoxin genes was of interest. A preliminary analysis of the genomesuggested that C. histolyticum possessed a single ORF that sharessignificant amino acid homology with C. septicum alpha toxin asdetermined by BLAST analysis of two databases. Therefore, the putativealpha toxin gene within C. histolyticum has been assigned as a homologof C. septicum alpha toxin. Studied extensively by the Rodney K. Twetenlaboratory, C. septicum alpha toxin was classified as a member of theaerolysin-like family of toxins. Notably, C. septicum alpha toxin doespossess haemolytic activity (Ballard et al. (1992), Infect Immun 60:784-790; Melton-Witt et al. (2006), Biochem 45: 14347-14354) and isdistinct from oxygen labile hemolysins as described for C. histolyticumε toxin (Hatheway (1990), Clin Microbiol Rev 3:66-98).

The C. septicum alpha toxin is elaborated as an inactive preproproteinwhich is processed to the extracellular environment as an inactiveprotoxin. The protoxin then binds to receptors on the cell membranewhere they are cleaved into their active structures by proteases(usually furin). A furin consensus site within the toxin is essentialfor activation by eukaryotic proteases. The activation involves thecleavage of 40-45 amino acids from the C-terminus. Absent the C-terminalcleavage the C. septicum alpha toxin is not functional. Full length C.septicum alpha toxin is haemolytic (Ballard et al. 1992). The activetoxin is approximately 41.3 kDa (Gordon et al. 1997). Once activated,the toxins oligomerize on the cell surface into a prepore complexfollowed by insertion of a beta-barrel into the membrane.

The model C. septicum alpha toxin consists of three distinct domainstermed: D1, D2, and D3. The D1 domain is involved with receptor bindingand oligomerization, while the D2 domain contributes toamphipathic-hairpin structure. The D3 domain has a D3 propeptide regionthat includes a short carboxyl-terminal peptide cleaved at the known ATactivation site (R398) and functions as an intramolecular chaperone thatprevents premature oligomerization of the alpha toxin. Using saturationmutagenesis, single amino acid substitutions within each domain haveallowed the determination of those residues essential for biologicalactivity (Melton-Witt et al., 2006). Importantly, the functional assayutilized a cell viability assay to determine LD₅₀ doses. Thus, therelative effect of single amino acid substitutions within the entirecoding region was assessed using a functional assay.

To further understand the primary structure of the CLH alpha toxin, theprotein alignment, performed in MATLAB, of the model protein (C.septicum alpha toxin) was made with the CLH alpha toxin. The results arepresented in FIG. 1.

The translated CLH putative alpha toxin has an identifiable signalsequence and has a very high probability of being a secreted protein.Thus, the first criterion of an exosubstance is achieved. There is a 75%positive homology between the C. septicum alpha toxin protein sequenceand the CLH alpha toxin protein sequence. Multiple regions of highhomology were identified between the model alpha toxin and the CLHputative alpha toxin. Such regions and essential amino acid residues arehighlighted in green shading in FIG. 1.

Notably, the alignment shows multiple differences in essential aminoacid residues that, based on the work of Melton-Witt et al. (2006)(Biochem 45:14347-14354), individually render the CLH_2834 & 2835protein non-functional. Beginning with the N-terminal region of themature protein, a 17 amino acid sequence region is missing in the CLHalpha toxin sequence which is located about 20 amino acids downstreamfrom the putative signal peptide cleavage site. Within this 17 aminoacid stretch, a W74 residue on C. septicum alpha toxin has beenidentified as a critical residue in loop 1 (L1). The lack of 17 aminoacids from the D1 domain in the CLH sequence version suggests an alteredstructure for this domain relative to a wild type and a disruption ofthe receptor binding functionality.

Within the C-terminal region of the protein, several amino acid residuechanges also render the CLH protein non-functional. The amino acid T302in the C. septicum alpha toxin was replaced by Proline in the CLH alphatoxin. Residue E303 in the C. septicum alpha toxin is replaced byThreonine in the CLH alpha toxin. The studies of Melton-Witt et al.(2006) (Biochem 45: 14347-14354) indicated that each of thesemodifications will individually result in 0% lethality. Of note is thecomparison of the activation site, or furin cleavage site, between thetwo sequences. The C. septicum alpha toxin exhibits a furin consensuscleavage site beginning with K391 and terminating at R398. This regionfits the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg (SEQ ID NO:31), although the minimal cleavage sequence is Arg-X-X-Arg (SEQ ID NO:32). The CLH putative alpha toxin has a Glutamine residue instead ofArginine in the analogous R398 position. Thus, the C. septicumactivation site possesses the amino acid sequence, DKKRRGKRSVDS (SEQ IDNO: 26), with R398 identified as a critical residue. The CLH alpha toxinhomologous sequence in the D3 peptide is NTSST-EQNVEV (SEQ ID NO: 27);beginning with N367 of SEQ ID. NO. 8. Therefore, the putative C.histolyticum alpha toxin furin cleavage site appears to benon-functional, and this protein, even if expressed, could not beprocessed by contact with eukaryotic cells furin protease to generate afunctional toxin. The findings of the comparative amino acid sequenceanalysis are summarized in Table 3.

TABLE 3 Summary of Amino Acid Sequence Alignment Comparison for PutativeCLH alpha toxin Protein Effect on Function C. septicum α toxin CLH 2834& 2835 Essential Amino Acid Residue W 74 Missing Receptor bindingdisrupted T302 P Lack of lethality E303 T Lack of lethality K391-R398T - - - Q Incapable of activation

The summary of the sequence alignment analysis suggests that theputative CLH alpha toxin possess a significant number of amino acidresidues differences that would make the mature protein non-functional.The phenotypic linkage to functionality for alpha toxin is thedemonstration of haemolytic activity. Importantly, the CollagenaseClostridium Histolyticum production strain does not exhibit haemolyticactivity when plated on blood agar. The results of a Blood agarhemolytic assessment are illustrated in FIG. 2.

Panel A of FIG. 2 shows the results obtained when a sample of C.histolyticum Clone 004 cell expansion is cultivated on Blood agar. Thereis no evidence of any beta hemolytic phenotype. In contrast, panel B ofFIG. 2 shows the results obtained when a sample of C. septicum iscultivated on Blood agar. There is clear evidence of beta hemolysis thatextends well beyond the area of sample application as indicated in PanelC. The images presented do not adequately represent the qualitativedifference observed when one views the test articles. The appearance ofbeta hemolysis is easily discernable and the complete lack of anyhemolysis in the C. histolyticum plate stands in stark contrast to thebroad zone of hemolysis noted when the C. septicum culture (producer ofα toxin) is inspected.

2. Delta (δ) Toxin

Hatheway et al. (1990) (Clin Microbiol Rev 3: 66-98) has defined the δtoxin of C. histolyticum as an elastase, primarily based on the initialresearch communication by Takahashi, et al. (1970) (BBRC 39: 1058-1064).No further substantial studies on this toxin have apparently beenpublished since then. Four fractions demonstrating elastase activitywere isolated from C. histolyticum by Takahashi et al. usingdifferential ultrafiltration. The primary focus was on a fraction whichpassed through membranes of nominal 50 kDa cut-off membranes but wasretained by membranes with a nominal 10 kDa cut-off.

Thermolysin is a zinc metalloprotease with a mature enzyme molecularweight of 34.6 kDa. Importantly, thermolysin is a model protein for aclass of proteins that contain a presequence employed in secretion(signal peptide) but also a lengthy prosequence of approximately 200amino acid residues that is two thirds the size of the mature protein.Thermolysin-like enzymes are elaborated as inactive preproproteins withthe prosequence serving a role as an inhibitor of the mature enzyme andalso as a chaperone to ensure proper folding of the enzyme (O'Donohue etal. (1996), JBC 271:26477-26481). The prosequence is autocatalyticallyremoved by the mature enzyme portion of the molecule in theextracellular environment. Thus, the maturation pathway forthermolysin-like enzymes includes: a secretion step, the presence of apro-mature form in the extracellular matrix, the cleavage of theprosequence, and the presence of a mature, active enzyme.

The gene sequence alignment for thermolysin and CLH_2576, the putativeC. histolyticum delta toxin, is illustrated in FIG. 3. This imagedisplays the full length prepromature amino acid sequence as a singleunit that is theoretically transcribed as a single polypeptide. Theinitial 28 amino acids at the N-terminus of thermolysin are shownjuxtaposed to the green shaded prosequence which terminates at Ser232.The unshaded mature amino acid sequence begins with Ile233. Using theSignalP program, the thermolysin and the CLH_2576 polypeptides arepredicted to be secreted. The translated putative C. histolyticum deltatoxin has an identifiable signal sequence and a very high probability ofbeing a secreted protein. There is a 65% positive homology between thethermolysin protein sequence and the CLH delta toxin protein sequence.

To understand the nature of the pro and mature forms of both proteins,the individual regions were analyzed as distinct sequences with regardsto functionality. The prosequence alignment is depicted in FIG. 4. Thereis a 57% positive homology between the two prosequence forms. A recentreview of the primary structural analysis of the prosequences of over100 thermolysin-like proteases was conducted by Demidyuk et al. (2008)(Protein J 27: 343-354). These investigators noted that considerablevariability existed within the prosequences, alternatively termedprecursors or propeptides. The prosequences were more tolerant tomutations compared to the corresponding mature enzymes. Nevertheless,regions exhibiting a high degree of conservation and substitutions inkey residues were noted which may dramatically alter the function. Theresidues shaded green in FIG. 3 identify those amino acid residues thatare critical for the prosequence to function. No differences are notedbetween the thermolysin and CLH_2756 sequences. Two residuescorresponding to Ile183 and Arg184 in the thermolysin sequence areshaded yellow; however, the substitutions in the CLH_2756 sequence aresimilar amino acids that likely do not result in any alteration offunction.

Importantly, there is a region of non-homology at the C-terminus of theprosequences as illustrated by the yellow shading of the CLH_2756sequence beginning with Ser185. This region is the site of autocatalysisand suggests that the CLH_2756 sequence is not an acceptable substratefor cleavage by the active site of the mature enzyme. The criticality ofthe amino acid residues around the cleavage site was investigated byWetmore et al. (1994) (Mol Microbiol 12:747-759), using Bacillus cereusthermolysin-like neutral protease as the model enzyme. Theseinvestigators determined that the processing was particularly sensitiveto the nature of the amino acid three residues upstream from thecleavage site. A consensus sequence was identified for the sequencearound the proprotein processing site and alterations in key residuesresulted in the non-export or nonprocessing of the protein to a mature,functional enzyme. Key features of the consensus sequence were: thepresence of a non-polar residue in position P₃ (Gly, Ala, Ile, Leu, orVal), a polar residue or Pro in position P₁ (Pro, Ser, His, Glu), and anon-polar residue in position P₁′. Additionally, the prothermolysinmaturation has been shown to occur between a serine and isoleucineresidue (O'Donohue et al. (1994), Biochem J. 300: 599-603). To explorethe sequence alignment around the cleavage site, a comparative sequenceassessment of the proprotein processing sites for thermolysin and forCLH_2576 can be made by inspection. It is apparent that the CLH_2576amino acid sequence in the proprotein processing area does not containthe appropriate amino acid arrangement to allow autocatalysis. When oneconducts a theoretical exercise to interrogate the CLH_2576 aminosequence to determine if the proprotein processing site is reasonablyclose to the predicted site based on sequence alignment, it is clearthat no adjustment allows the proper amino acid sequence to beidentified. Shifting the proprotein processing site 2 residues to theC-terminal side allows for the proper arrangement of amino acids that donot violate the Wetmore et al. rules. However, the Ser-Ile rule ofO'Donohue et al. (1994) (Biochem J. 300: 599-603) is not present. Thus,it is concluded that the proprotein form of the CLH_2576 polypeptide isnot a suitable substrate for autocatalysis. The net effect is that themature, active enzyme is not present in the cell broth of C.histolyticum (Clone 004).

To explore the mature forms of both proteins, the comparative sequencealignment is depicted in FIG. 5. An inspection of the sequence alignmentin FIG. 5 suggests that many essential amino acids have been conserved.Notably, the AHELTHAVTD sequence (SEQ ID NO: 28) of the MatureThermolysin, beginning with Ala140 of SEQ. ID. NO. 13 has beenidentified as a component of the active site for thermolysin and thehigh homology displayed by CLH_2576 of SEQ ID. NO. 14 suggests that CLHdelta toxin is a member of the thermolysin class of proteases (Kooi etal. (1996), J Med Microbiol 45:219-225; Kooi, et al., (1997), InfectImmun 65:472-477). Multiple residues shaded in green have beenidentified as essential for binding or catalysis. One notable differencebetween the sequences of the two molecules is the GGI region beginningwith G135 in thermolysin. This stretch of amino acid residues is highlyconserved in thermolysin-like proteases with no defined functionassigned (Frigerio et al. (1997), Protein Eng 10:223-230). Thecorresponding CLH_2576 region possesses several significant differencesin this sequence. Nevertheless, the overall high degree of homology andthe conservation of essential amino acid residues confirm the selectionof CLH_2576 as delta toxin with predicted molecular mass ofapproximately 35 kDa. This assessment aligns with the informationpresented by Takahashi et al (1970) (BBRC 39: 1058-1064).

In summary, the putative CLH delta toxin has been identified usinggenome sequence analysis. However, the interrogation of this sequencesuggests that the cleavage of the proprotein will not occur, renderingthis molecule non-functional. Therefore, it is deduced that the δ toxin,if expressed and secreted in the Clone 004 derivative of C. histolyticumATCC 21000, is not functional.

3. Epsilon (ε) Toxin

MacLennan et al. (1962) (Bact Rev 26:176-274) and Hatheway described theε toxin of C. histolyticum as an oxygen-labile haemolysin serologicallysimilar to those produced by other strains of Clostridium, such as C.tetani, C. novyi, and C. septicum. Bowen (1952) (Yale J Biol Med25:124-138) demonstrated that the ε toxin was expressed during theexponential phase and degraded during the stationary phase as observedfor the α toxin activity, and was similarly degraded by proteinases invitro.

An inspection of the BLAST analysis results of the C. histolyticumgenome identified an ORF coding for a hemolysin that was in the sameclass as perfringolysin and tetanolysin, which are members ofthiol-activated, pore forming proteins with affinity for cholesterol.Such proteins are part of a family of Cholesterol Dependent Cytolysins(CDC) and all exhibit distinctive protein sequences and uniquestructures. Over 25 CDC proteins have been identified with completeprotein sequences available. The CDCs are a group of β-barrelpore-forming toxins secreted by various species of Gram positivebacteria all in the 50-60 kDa molecular weight range. The prototypicalCDC is perfringolysin which serves as a model protein for all CDCs(Heuck et al. 2007, JBC 282: 22629-22637). The typical organization of aCDC includes a cleavable signal sequence to facilitate the exports tothe extracellular environment as a water-soluble monomeric protein.Subsequently, the folded monomeric form binds to a target eukaryoticmembrane, mediated by cholesterol binding, and then oligomerizes on themembrane surface to form arcs and ring-like structures that areresponsible for the cytolysis. The CDCs are also known asthiol-activated cytolysins and were originally described as hemolysins(Billington et al., 2000).

The gene sequence alignment for perfringolysin and CLH_1920, theputative epsilon toxin, is illustrated in FIG. 6. This image displaysthe full length (pre plus mature) protein sequence as a single unit thatis theoretically transcribed as a single polypeptide. The initial 29amino acids at the N-terminus of perfringolysin are illustrated with ablue star above Lys 29 at the site of signal peptidase cleavage. TheSignalP analysis of the CLH_1920 sequence did not identify arecognizable signal peptide cleavage site and was predicted to be anon-secreted protein. There is an 84% positive homology between theperfringolysin protein sequence and the CLH_1920 putative epsilon toxinprotein sequence.

The amino acid residues shaded in green denote essential amino acidsthat are conserved between the two proteins. Importantly, the 11 aminoacid sequence ECTGLAWEWWR (SEQ ID NO: 29), beginning with glutamine 458of SEQ. ID. NO. 15, is an essential region that is termed theundecapeptide sequence. Along with the high degree of homology withinthe sequence designated as the mature protein region, this undecapeptidesequence serves to identify the CLH_1920 protein as a CDC. Therefore,the CLH_1920 protein, if elaborated as a secreted protein, would beexpected to have haemolytic functionality. A single region ofnon-homology between the two proteins is highlighted in yellow shading.Importantly, the C-terminus of CDCs has been shown to be critical forcholesterol binding (Shimada et al., 1999, JBC 274: 18536-18542). Theprocess of hemolysis by CDCs involves two critical steps prior to poreformation: binding and membrane insertion. Shimada, et al. (1999) (JBC274: 18536-18542) demonstrated that modest changes to the C-terminusaffected the binding step. An alteration of the 3′ terminal amino acidsseverely reduces cholesterol binding as measured by an ELISA method. Thecorresponding haemolytic activity on red blood cells was coordinatelyreduced or eliminated depending upon the severity of the C-terminalamino acid change. An inspection of the C-terminus of the CLH_1920sequence shows some significant differences compared to theperfringolysin sequence.

As summarized in Table 4, the haemolytic activity of the putative C.histolyticum epsilon toxin may be absent due to two features of thetheoretical amino acid sequence. First, the molecule is predicted not besecreted; thus, the molecule would not be available for interaction withtarget cells. Second, the C-terminus of CLH_1920 protein does notpossess a homologous region for cholesterol binding, which suggests thatan important element associated with hemolysis may be defective.

TABLE 4 Summary of Amino Acid Sequence Alignment Comparison for PutativeCLH epsilon toxin Protein Effect on Function Perfringolysin CLH 1920Region Characteristic N-terminal Missing signal Not secreted peptidasecleavage sequence C-terminal Non-consensus Lack of cholesterolbinding/no activation

Non-clinical toxicity studies demonstrated no clinical and morphologicalindications of hemolysin effects in vivo. The data generated by localand IV bolus administration support the absence of haemolytic toxinssuch as ε toxin.

The absence of haemolytic toxins can be verified by the plating of testmaterial on blood agar which is routinely performed at the end of eachC. histolyticum Clone 004 fermentation, which also confirms the absenceof foreign growth. The expression of haemolytic toxins results in thelysis of the blood cells, and thereby resulting in the formation ofdistinct halos around colonies producing haemolysins. The Collagenase C.histolyticum production strain does not produce halos or zones ofclearance (see FIG. 2) supporting the absence of ε toxin and any otherhaemolytic entities in the production strain. To verify the hemolyticfunction of a CDC, commercially available tetanolysin was applied toBlood agar to mimic the routine plating test. The results areillustrated in FIG. 7 which shows the beta hemolytic phenotype observedwhen 10 μcL of a 10 μg/mL solution of tetanolysin in phosphate bufferedsaline is applied to the surface of Blood agar, then incubated for 24hours at 37° C. Thus, if a functional CDC were present in the testmaterial, the beta hemolytic phenotype should be observed.

4. Clostripain or Gamma (γ)-Toxin

The gamma toxin of C. histolyticum has been described as clostripain, acysteine endopeptidase (EC 3.4.22.8). Dargatz, et al. (1993) (Mol GenGenet 240:140-145) cloned and sequenced the C. histolyticum gene forclostripain and this information was deposited in GenBank underaccession number X63673 (www.ncbi.nlm.nih.gov/nuccore/X63673.1). Tounderstand the primary structure of the CLH_1861 gamma toxin, theprotein sequence alignment from MATLAB of the model protein (C.histolyticum clostripain) was made with the CLH_1861 gamma toxintheoretical sequence. The results are presented in FIG. 8.

An inspection of FIG. 8 shows a very high degree of homology (99%)between the model clostripain and the sequence obtained from the genomeanalysis. In fact, there are only 3 amino acid differences, none ofwhich are residues identified as essential for activity. Those criticalamino acids identified in literature studies as essential forfunctionality are shown in green shading. SignalP analysis of bothproteins indicated that high secretion score and the signal cleavagesite depicted with a blue star (Labrou et al. (2004). Eur J Biochem271:983-992). Thus, one would predict that the CLH_1861 molecule wouldbe secreted and functional. A residual clostripain analysis wasconducted as part of routine release.

The clostripain analysis supports the merits of the sequence alignmentapproach for the C. histolyticum toxins in general. One would predictthat the presence of a functional toxin gene would necessarily translateinto an amino acid sequence that shared a high degree of homology with aknown model protein. Further, the conservation of essential amino acidresidues would also be a characteristic of a functional toxin gene.

The information obtained from the genome sequence analysis providedevidence that loci for putative alpha, delta, and epsilon toxins werepresent. Further analysis of the theoretical primary structure of eachtoxin indicated that non-functional forms of each toxin were predictedas a consequence of key defects in the amino acid sequence of eachtoxin. Notably, the alpha and epsilon toxins can be assigned ashomologues to two classes of pore-forming, hemolytic molecules. As theend of fermentation, samples from every batch are plated onto blood agaras part of a routine purity test. The lack of halos or zones ofclearance around the colonies confirm the absence of haemolytic activityin the culture and fermentation. Consequently, the absence of haemolytichalos around the end of fermentation samples demonstrate the absence ofboth a and ε toxins on a continuing basis.

Table 5 shows the results from the sequence analysis and predictedfunctionality. The results confirm why Clone 004 has functionally shownthe absences of toxicity and the lack haemolytic activity.

TABLE 5 Summary - Predicted Status of C. histolyticum Clone 4Exosubstances Predicted Toxin CLH Name Sequence Result Functionalityalpha CLH_2834 & Missing critical aa Non functional; 2835 residuescorrelated through absence of haemolytic activity on blood agar platesdelta CLH_2576 Missing consensus Non-functional proprotein cleavagesequence epsilon CLH_1920 Signal peptidase Not secreted, non- cleavagesite functional correlated defective & non- through absence of consensushaemolytic activity on cholesterol blood agar plates binding sequencegamma CLH_1861 Clostripain Functional

5. C. histolyticum Sequence Analysis of Beta Toxins (Collagenase I andCollagenase II)

The sequence analysis of the putative C. histolyticum beta toxin loci ispresented in FIGS. 9 and 10. As shown in FIG. 9, the amino acid sequenceof the mature collagenase I of clone 004 (CLH_1768 and 1769; SEQ ID NO:3) differs from the translated colG sequence (SEQ ID NO: 19) by threeamino acids. FIG. 10 shows that the amino acid sequence of the maturecollagenase II of clone 004 (CLH_2116; SEQ ID NO: 4) differs from thetranslated colH sequence (SEQ ID NO: 20) by eight amino acids. Bothcollagenases are fully functional.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by references. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

REFERENCES

-   1. Nielsen et al. (2004) In J. Glasgow et al., eds., Proc. Sixth    Int. Conf. on Intelligent Systems for Molecular Biology, 122-130.    AAAI Press, 1998.-   2. Hatheway (1990) Clin Microbiol Rev 3:66-98.-   3. Ballard et al. (1992) Infect Immun 60:784-790.-   4. Melton-Witt et al. (2006) Biochem 45:14347-14354.-   5. Gordon et al. (1997) Infect immun 65:4130-4134.-   6. Takahashi et al. (1970) BBRC 39:1058-1064.-   7. O'Donohue & Beaumont (1996) JBC 271:26477-26481.-   8. Demidyuk et al. (2008) Protein J 27:343-354.-   9. Wetmore et al. (1994) Mol Microbiol 12:747-759.-   10. O'Donohue et al. (1994) Biochem J. 300:599-603.-   11. Kooi & Sokol (1996) J Med Microbiol 45:219-225.-   12. Kooi et al. (1997) Infect Immun 65:472-477.-   13. Frigerio et al. (1997) Protein Eng 10:223-230.-   14. MacLennan (1962) Bact Rev 26:176-274.-   15. Bowen (1952) Yale J Biol Med 25:124-138.-   16. Heuck et al. (2007) JBC 282:22629-22637.-   17. Billington et al. (2000) FEMS Microbiol Lett 182:197-205.-   18. Shimada et al. (1999) JBC 274:18536-18542-   19. Dargatz et al. (1993) Mol Gen Genet 240:140-145.-   20. Labrou & Rigden (2004) Eur J Biochem 271:983-992.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A recombinant nucleic acid molecule, comprising:a) a polynucleotide having the nucleic acid sequence of SEQ ID NO: 2 orthe complement of SEQ ID NO: 2, and b) a heterologous regulatorysequence operably linked to the polynucleotide.
 2. The recombinantnucleic acid molecule of claim 1, wherein the heterologous regulatorysequence is a heterologous promoter operably linked to the nucleic acidsequence of SEQ ID NO: 2 or to the complement of SEQ ID NO:
 2. 3. Avector comprising the nucleic acid molecule of claim
 1. 4. The vector ofclaim 3, wherein the vector is a plasmid.
 5. A recombinant host cell,comprising the vector of claim
 3. 6. The recombinant host cell of claim5, wherein the host cell is selected from the group consisting of abacterial cell, a fungal cell, an insect cell, a plant cell and amammalian cell.
 7. The recombinant host cell of claim 6, wherein thehost cell is selected from the group consisting of E. coli,Streptomyces, Pseudomonas, Serratia marcescens, Salmonella typhimurium,a yeast cell, plant cells, thymocytes, Chinese hamster ovary cell (CHO),COS cell, and Lactococcus lacti.
 8. The recombinant nucleic acidmolecule of claim 1, wherein the recombinant nucleic acid molecule isfused to a marker sequence.
 9. The recombinant nucleic acid molecule ofclaim 8, wherein the marker sequence encodes a polypeptide selected fromthe group consisting of: a glutathione-S-transferase (GST) fusionprotein, a hemagglutinin A (HA) polypeptide marker from influenza, andhexa-histidine peptide.
 10. The vector of claim 3, wherein the vector isintroduced into a host cell using conventional transformation ortransfection techniques.
 11. The recombinant nucleic acid molecule ofclaim 1 having a biological activity of a naturally-occurringcollagenase.